As secondary active transporters, CLCs harness energy stored in one ion gradient (Cl- or H+) to pump the other ion against its electrochemical gradient. This occurs through tight coupling of protein conformational changes to ion binding, unbinding, and translocation events. The X-ray crystallographic structures of several CLC homologs provide an invaluable structural foundation for understanding molecular mechanisms in this family. Despite extensive efforts, however, crystallization has revealed only one basic CLC conformational state. In this project, we will characterize the unknown conformational states and describe both the dynamics within these conformational states as well as the structural transitions between the states, which together give rise to CLC transporter function. In Aim 1, we will characterize the molecular details of the elusive outward-facing (OF) and inward-facing (IF) states by using double electron-electron resonance (DEER) to measure distance changes between pairs of site- directed spin labels on CLC-ec1, a well-studied homolog for which the structure of the occluded state has been determined. Computational modeling will guide DEER experimental design, and experimental results will guide the refinement of the structural models and validate modeling predictions. Crystallization of states will be approached using state-stabilizing cross-links designed based on the structural models, as well as conformation-specific synthetic antigen binders that can be used as crystallization chaperones. In Aim 2, we will investigate three aspects of conformational dynamics within and between states that are key to elaborating the overall CLC transport mechanism. First, we will characterize how water dynamics and H+- transport pathways vary amongst the different conformational states using extended molecular dynamics (MD) simulations combined with experimental validation. Second, we will identify potential intermediates in the transport cycle and evaluate kinetics of transitions between states using single-molecule fluorescence resonance energy transfer (smFRET) measurements and rapid-freeze quench DEER. Finally, to investigate in detail how binding and translocation of ions are coupled to protein conformational changes, we will model transitions between the states. Advanced non-equilibrium simulations and sampling techniques will be used to describe the transition pathways between the major states (IF, OF, and occluded) and to calculate the free energy profiles associated with these transitions.